Defects in the hematopoietic microenvironment, associated with the Steel (Sl) mutation in mice, have been shown to be due to abnormalities in the production or presentation of the protein product of the Steel gene.

This product is termed stem cell factor (SCF) or mast cell growth factor (MGF). It exists as a locally secreted or membrane-bound protein.

Stable stromal cell transfectants can differentially process the two forms of human SCF protein product, membrane-bound and secreted SCF.

Membrane-bound SCF is available in the transfected Sl/Sl4 cell line, Sl/Sl4 hSCF220 (ATCC CRL-2453).

1. Check all containers for leakage or breakage.

2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below --130°C, preferably in liquid nitrogen vapor, until ready for use.

1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).

2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.

3. Transfer the vial contents to a 75 cm2 tissue culture flask coated with 0.1% gelatin and dilute with the recommended complete culture medium (see the specific batch information for the recommended dilution ratio).   It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).

4. Incubate the culture at 37°C in a suitable incubator.  A 5% CO2 in air atmosphere is recommended if using the medium described on this product sheet.

Note: If it is desired that the cryoprotective agent be removed immediately, or that a more concentrated cell suspension be obtained, centrifuge the cell suspension at approximately 125 x g for 5 to 10 minutes.  Discard the supernatant and resuspend the cells with fresh growth medium at the dilution ratio recommended in the specific batch information.

1. Remove and discard culture medium.

2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.

3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.

4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.

5. Add appropriate aliquots of the cell suspension to new culture vessels coated with 0.1% gelatin.

6. Incubate cultures at 37°C.

Subcultivation Ratio: 1:10 to 1:30
Medium Renewal: Every 2 to 3 days

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.